Process of making a nutrient material



- Patented Nov.- .28, 1944 2,364,008 rnocass or MAKING A NUTRIENTMATERIAL Elmer H. Stuart, Indianapolis, Ind., assignoi' to Eli Lilly andCompany, Indianapolis, hit, a corporation oi Indiana No Drawing.

1 .Claim.

This invention relates to nutrient substances and more particularly tonutrient materials suitable for oral, rectal, and intravenousadministration.

An object of this invention is to provide a-material which supplies thenecessary nitrogenous materials required for the sustenance of life,which is readily assimilated, which is non-toxic, and which iseconomical to prepare.

It has been well established that approximately nine or ten amino acidsin the presence of other amino acids comprise the necessary nitrogenousmaterials required for the maintenance of life. (The Chemistry oftheAmino Acids and Proteins, by Carl L. A. Schmidt, published by CharlesC. Thomas, 1938, page 986.) These amino acids are usually derived fromproteins, and ordinarily the human or animal would be adequatelysupplied with these essential amino acids from foods which arecustomarily consumed. However, .whensituations arise which interferewith normal digestion, it is necessary to supply the required elementsfor sustenance by parenteral administration. For this purpose mixturesof amino acids have heretofore been proposed. A review of the literaturereveals the unanimous belief that the digestion of the protein must besuch that the protein is broken down into free amino acids. Inconformity with this belief, the nutrient material for the supply ofnitrogenous material required for the sustenance of life heretofore usedintravenously contained essentially free amino acids obtained by thecomplete digestion of a protein material. (Journal of the AmericanMedical Assn., vol. 112, No. 9, pp. 796-802.) These mixtures of freeamino acids, however, have been found to be relatively unsatisfactory.

In accordance with this invention, a nutrient .materlal is providedwhich supplies all of the nitrogenous materials required for themaintenance of life and which is markedly less toxic than the mixturesof amino acids heretofore employed. This nutrient material is suitablefor oral, rectal, and parenteral administration and more particularlyfor intravenous administration; A group of rabbits was given two gramseach of the nutrient material of this invention daily and a similargroup was supplied with an equal quantity of mixtures of free aminoacids prepared by the complete digestion of proteins with pancreaticenzymes. Both compositions were administered intravenously and therabbits received no further nutrient material other than glucose andwater. The dail loss of weight in Application September 22, 1940, SerialNo. 358,037

the rabbits treated with the nutrient material of this invention wasabout one half of that of rabbits supplied with the mixture of freeamino acids. For a 14-day period, in many cases those treated with thefree amino acids died, while in the vast majority of cases the rabbitssupplied with the nutrient material of this invention survived.

The nutrient material of this invention comprises polypeptides which aresuitable for oral, rectal, and parenteral administration.- These-polypeptides are water-so1uble,-are not precipitated by chloraceticacid, and give a positive biuret reaction. Amino acids give a negativebiuret reaction. The color obtained in the 'bluret test'with thepolypeptides is somewhat diiferent from that obtained from the proteinsfrom which the polypeptides are derived in that the polypeptides yield acharacteristic pink color, whereas the original proteins give ablueviolet colored biuret reaction. The polypeptides containchemically-combined amino acids, can be obtained most economically fromnatural proteins, and are markedly less toxic and more readily solublein water than are the free amino acids which are chemicall combined inthe polypeptides. The polypeptides of this invention are soluble inwater having a hydrogen ion concentration of pH 6. The polypeptides ofthis invention are prepared by digesting a protein in a medium having apH between 4 and 5 with a proteolytic enzyme; such as papain, or thoseoccurring in liver, kidney, and other animal tissue. However, it ispreferred to employ papain. The protein is desirably a natural protein,such as casein, the protein from soy bean, 'or certain animal tissues.Preferably, the protein or plurality of proteins employed for preparingthe polypeptides contains all of the amino acids required for the supplyof the necessary nitrogenous materials for the sustenance of life. Therate .at which the digestion proceeds is dependent upon the temperature.Most proteolytic enzymes operate most satisfactorily at about 37 C.Temperatures below 37 C. require a much longer period of digestion. Inthe case of papain, the rate of digestion may be markedly increased byusing temperatures as high as C. This digestion is continued desirablyuntil the amount of free carboxyl groups in one gram of the drieddigested material is equivalent to between 0.075 and 0.2- gram of sodiumhydroxide. The free carboi'rylic group is determined by the methoddescribed in Allens Commercial Organlc Analysis, fifth edition,published by P. Blaklstons Son 8: 00., Inc., 1930, Pp. 727-729, in

the modification in which 85% acetone is used instead. of alcohol. Thedigested mixture is then heated to about 100 C. to coagulate undigestedproteins; and after coagulation the insoluble proteins are removed fromthe mixture by any suit-' able means, such as decantation, filtration,or centrifugation. If the digestion of the protein is permitted toproceed so that one gram of the material neutralizes an amount of sodiumhy- Alternatively, the digestion of the protein in accordance with thisinvention may be continneed until the a-amino nitrogen is between 0.5%

and 3.0% of the dried product as determinedby the Van Slyke methoddescribed in Comp. rend. du Lab. Carlsberg, Ser. Cjhim., vol. 22, pp.480- 486.

After the insoluble proteins have been removed, the digested mixture maybe used as a nutrient material. This nutrient material is notprecipitated by chloracetic acid, and if desired the digestion may becontrolled by this test although the two tests heretofore described arepreferred.

If desired, the digested material may be evaporated to dryness and theresidue may be redissolved for administration orally, rectally, orparenterally. However, it is preferred that the digested mixture befurther refined by a re digestion process. To produce these improvedpolypeptides, the digested mixture, after removal of the insolubleproteins, is adjusted to a pH of about 4.5 and is then redigested with aproteolytic enzyme, such as papain,-until the pH of the solution hasreached a constant value. Preferably, the temperature is maintained at37 -C., although, as heretofore stated, the digestion may be practicedat other temperatures. It is then heated to about 100 C. to destroy theproteolytic enzymes. The insoluble material is desirably removed. Theresulting solution may be used in that .form or evaporated to drynessand redissolved. Animals to which this refined product is administeredintravenously lose less weight over a period of time than thosereceiving the unredigested product.

Many proteins, such as casein; are known which contain all of the aminoacids required for the maintenance of life in humans and animals.Accordingly, to produce a nutrient material comprising primarily aplurality of polypeptides which contain these amino acids chemicallycombined, such protein is digested in accordance with the methodheretofore outlined. In the event that a protein is deficient in aparticular retrate and pounds of commercial casein. (The sodium citrateis employed as a buffer salt.) Four pounds of papain, which has firstbeen dissolved in water, are added together with 2.5 quarts of tolueneas a preservative. is then closed and 2 lbs. 14 oz. of hydrogen sulfideare introduced into the solution, while the solution is gently agitatedin the closed kettle. This quantity of hydrogen sulfide is usuallysufiicient to reduce the pH to about 4.5. Desirably, the mixture istested at this point to insure that the pH is between 4 and 5. Themixture is allowed to stand for about two hours and then graduallywarmed during about 24 hours to between 40 and 50 C. Thereafter it isstirred twice daily until such time as a sample indicates that the freecarboxyl groups in one gram of the dried material are equivalent tobetween 0.075 g. and 0.2 g. of sodium hydroxide, or the a-amino nitrogenis between 0.50% and 3.0% of the dried material. This procedure usuallyrequires from five to ten days, depending on the temperature at whichthe material is digested and during this time the casein is essentiallybroken down into polypeptides. The mixture is then heated to boiling fora period of 10 to 15 minutes, cooled to about room temperature, andfiltered. The filtrate is returned to the kettle, and one pound ofpapain and 2.5 quarts of toluene are added. Sufficient hydrogen sulfideis added to reduce the pH to about 4.5. Usually about one pound ofhydrogen sulfide is required for this purpose. The solution is digestedat about 37 C. until such time as the pH has reached a constant value.This procedure requires from six to ten days. It has been found thatthis final pH varies according to the type of protein. materialused, butin this case, which is casein, it is approximately 5; in the case of theprotein of soy bean, it is approximately 5.8;,in the case of the proteinof liver it is approximately 6.3. The mixture is then heated to boilingto destroy the enzymes, cooled, filtered, and the filtrate evaporatedtodryness in vacuum. The dry material obtained from the filtrate whichcontains the polypeptides has total nitrogen 11.68%, a-amino nitrogen1.95%, free carboxyl groups in one gram equivalent to 0.16 g. sodiumhydroxide, and gives a characteristic pink color biuret reaction. Thepolypeptides are soluble in a water solution having a.pH of 6.

Example 2.Nutrient material obtained from the protein of soy bean.

To 10,000 cc. of water are added in a digestion kettle 475 g. offat-free soy bean meal, 25 cc. of toluene, 20 g. of papain, and 12 g. ofhydrogen sulfide. The top of the kettle is closed and the mixture ismaintained at 50 C. until the free carboxyl groups in one gram of thedried material are equivalent to between 0.075 g. and 0.2 g. of sodiumhydroxide or the a-amino nitrogen is between 0.5 and 3.0% of the driedmaterial. procedure usually requires a digestion of about one week. Themixture is boiled 15 minutes, cooled, and filtered. To the filtrate areadded 4 g. of papain, 25 cc: of toluene, and about 4g. of hydrogensulfide. This mixture is then digested at 37 C. until the pH attains aconstant value of about 5.85. The mixture isv then heated to boiling todestroy the enzymes, cooled, filtered, and the filtrate evaporated todryness in vacuum. The dry material obtained from the filtrate containstotal nitrogen 10.06%, a-amino nitrogen 3.15%, free carboxyl groups inone gram equivalent to 0.15% sodium hydroxide and gives a char-.

The kettle This' 2,864,008 acteristic pink color biuret reaction. Thesepoly-.

peptides are soluble in a water solution having a pH of 6.

Example 3'.-"-Nutrient material obtained from v the protein of liver.

To 1500 g. of fresh ground hog liver in a digestion kettle are added10,000 cc. of water contain- 1113 25 cc. of toluene and 15 g. ofhydrogen sulfide. (The enzyme is contained in the liver itself. Tofacilitate the digestion, if desired, 20 g. of papain may be added.) Themixture is digested at a temperature between 37 and 50 C.

1 until the free carboxyl groups in one gram of the dried material areequivalent to between 0.075 g. and 0.2 g. of sodium hydroxide, or thatthe the dried material; During this period, the protein in the hog liverisconverted substantially into polypeptides by the naturally occurringproteolytlc enzymes in the hog liver. The mixture. is heated to boiling,cooled, and filtered. To the filtrate are added 25 cc. or toluene, 4 g.of papain, and 5g. of hydrogen sulfide. The mixture is tained from thefiltrate contains total nitrogen 12.12%, a-amino nitrogen 2.08%. freecarboxyl' groups in one gram or the dried material equivalent to 0.16 g.sodium hydroxide and gives a characteristic pink color biuret reaction.The polypeptides are water-soluble in a solution having a pH 01' 6. 1

digested at about 37 C. until the pH attains a constant value. Themixture is heated to boiling, cooled, filtered, and the filtrateevaporated to dryness in vacuum. The dry material ob- While preferredembodiments of this invention .have been described, variousmodifications may be made therein without departing from' the scope ofthe appended claims.

What ls-claimed is:

The process of making a nutrient material suitable for oral, rectal, andparenteral administration, which consists in subjecting protein materialto digestion with papain in the presence of sumcient hydrogen sulfide toproduce a DH between 4 and 5, at a temperature between 37 and C. for aperiod of several days and until test of a sample removed and heated toboiling and filtered and dried indicates that one gram of the drymaterial .is neutralized by between 0.075

g. and 0.2 g. of sodium hydroxide, then heating to about C. to coagulateproteins, and then removing insoluble material.

ELM'ER H. STUART.

